The study of human cells for medical screening, diagnostic and other medical purposes is well known. For example, red blood cell count, mean cell volume (MCV), hemoglobin content and hematocrit are well known and commonly employed red blood cell parameters used in medical study and patient care.
The red blood cell is an excellent osometer in that the cell will change in shape and volume, depending on the osmotic pressure or chemical nature of the fluid surrounding it. If the red blood cell is suspended in a solution of the same osmotic pressure as that of the intracellular fluid, the suspending fluid is said to be isotonic. Should the red blood cell be suspended in a hypotonic suspending solution, the red blood cell will swell and may even rupture, due to the intake of water, in an effort to balance the internal osmotic pressure to that of the suspending solution. Other cells of the blood, namely the white blood cells and platelets, will act in much the same manner.
It is also well known that the osmotic pressure of solutions, i.e., their osmolality, such as saline solutions, varies with their concentration and types of solutes, in that the difference between the osmotic pressure within a cell and that of its suspension liquid causes the previously described change in volume and also a change in electrical resistance. In addition to a hypotonic solution, other volume changing solutions are known in the art. For instance, when red blood cells are exposed to hemolytic agents such as saponin, their membrane lipids are altered, so that water is allowed to enter the cell, causing the cell to swell and eventually rupture. If an excess of lytic agent is used, the red cell may be completely ruptured into extremely small fragments.
Cell and particle counting and measuring instruments, examples being those sold under the trademark Coulter Counter.RTM. by Coulter Electronics, Inc., Hialeah, Fla., employ electronic sensing means which directly respond to the electrical resistance of each cell to count and measure each cell and progressively record cell parameters of a sample of cells in an isotonic solution. The Coulter Counter.RTM. particle measuring instruments operate upon the well-known and documented principle of particle and cell measurement employing a sensing aperture path, which also is disclosed in Coulter U.S. Pat. No. 2,656,508 and improvement U.S. Pat. No. 3,259,842. The response of a Coulter Counter.RTM. electric sensor is influenced at least by the shape, deformability and flow rate of the microscopic item being measured as it flows through the sensing aperture path. Since cells are subject to some deformation as they pass through the sensing aperture path, their electrical resistance measurement and their measured volume may differ from their true volume. To distinguish between true volume and measured volume, the term "apparent volume" will be employed herein to refer to measured volume. It is also well-known that as the cells swell and their pores expand, the cell will be more conductive of the current so that its apparent volume will decrease with respect to its true volume.
In the commercial Coulter Counter.RTM. particle analyzer constructed in accordance with the heretofore mentioned U.S. Pat. No. 2,656,508, field excitation has been supplied by a direct current or low frequency source. As previously described, the electrical change caused by the passage of a particle through the electric field of small dimensions, excited by a direct or low frequency current, is approximately proportional to particle size. A direct current is considered to be of zero frequency in this application. However, the impedance sensing principle has been materially expanded to provide information concerning particles being studied, not limited only to characteristics due to the size of particles, but including characteristics due to the composition and nature of the material constituting the particles, as disclosed in U.S. Pat. No. 3,502,974 to Coulter et al and U.S. Pat. No. 3,502,973 to Coulter et al. These prior art apparatuses generally have at least two current sources, both of which are applied to the sensing zone simultaneously, one having a radio frequency and the other being a "zero frequency" direct current or, alternatively, having a sufficiently low frequency that the reactive part of the particle impedance has a negligible effect on the response of the apparatus. At radio frequencies, the high frequency current shunts the cell's membrane so that the high frequency measurement gives a size measurement which is a function of the cell's size and its internal conductance. One of the useful particle descriptors that can be obtained from this dual source arrangement is known in the art as the "opacity" of the particles. With a biological cell, opacity measures the internal conductivity of the cell. Opacity also can be described as measuring the ratio of size as measured at radio frequency to size as measured at low or zero frequency.
U.S. Pat. No. 3,836,849 to Coulter et al teaches that cells can be treated, for example, by a lysing agent to selectively cause the electronically measured opacity of different types of cells to change; whereby, each distinctive type of cell acquires a distinctive opacity range that is subject to electronic detection.
In U.S. patent application Ser. No. 118,727 to Shine, filed Feb. 5, 1980 and now U.S. Pat. No. 4,278,936 a method is described wherein biological cells are subjected to hypotonic solutions having different osmolalities. This causes the cells to rapidly attain a change in volume and electrical resistance parameters, which change is measurable by the above described Coulter Counter.RTM. particle analyzer.
In U.S. patent application Ser. No. 251,668, filed April 6, 1981, to Armstrong, now U.S. Pat. No. 4,374,644 there is disclosed a method wherein cells are subjected to a hypotonic solution or a solution having a lytic agent, causing the cells to attain a change in volume and electrical resistance parameters. This change is measured as a function of time by a Coulter Counter.RTM. particle analyzer.
In the article entitled "Erythrocyte Osmotic Fragility: Micromethod based on Resistive-particle Counting", by Adrian Gear, J. Lab. Clin. Med., Vol. 90, No. 5, pp. 914 (1977), there is described a method wherein cells in a hypotonic solution are subjected to higher than normal D.C. currents to obtain two--distinct peaks for intact and ruptured cells.
In the article entitled "High-Resolution Particle Analysis--Its Application to Platelet Counting and Suggestions for Further Application In Blood Cell Analysis", by John Hanes, Blood Cells 6, p. 201-213 (1980), there is described a method wherein numerous hypotonic solutions are prepared with cells therein and then the cells are put into a normal saline solution. When the MCV of the cells is then measured using a DC current in an electronic volume sensing particle analyzer, it is claimed that two distinct peaks are obtained for the intact and the ruptured cells. From this data the ratio or percent lysis is calculated at each ionic strength.
U.S. Pat. No. 4,278,936 to Shine and U.S. Pat. Nos. 3,502,973 and 3,502,974 to Coulter et al are incorporated by reference herein.